The peptide hormone relaxin (RLN) and its G protein-coupled receptor RXFP1 are expressed in several types of cancer cells, including prostate, endometrial, thyroid and others. It was shown that the overexpression of relaxin is often associated with advanced metastatic disease. Stimulation of RLN/RXFP1 signaling increases cell proliferation, invasion, migration, adhesion, and decreases cell apoptosis in vitro and in viv. RLN activates a set of signaling pathways and genes previously shown to be involved in tumorigenesis. More importantly, a suppression of RLN or RXFP1 by siRNA knockdown or through peptide antagonist expression in prostate cancer cells has an inhibitory effect on tumor growth and metastasis. This establishes the RLN signaling pathway as a promising novel anticancer target. To date no small molecule or non-peptide antagonists of the relaxin receptor are known. The current application is designed to fill this gap through high throughput screening (HTS) of a >400,000 small molecule compound library at NIH NCGC. An easily detectable and reliable indication of RXFP1 activation is an increase of cAMP production. Using HEK293T cells stably transfected with RXFP1 we have optimized a time-resolved fluorescence resonance energy transfer cAMP assay for quantitative HTS of RXFP1 antagonists in a 1536-well format. This assay will be applied for the RXFP1 antagonist screening campaign. After the primary screen the active compounds will be selected in a series of secondary assays designed to identify specific relaxin receptor antagonists. These include a counterscreen against cells transfected with related GPCR, cells stimulated with non-specific activator of cAMP, and a conformation screen using an orthogonal detection method. Tertiary cell-based assays will be used to select antagonists affecting RLN-induced responses in prostate cancer cells. Finally, we will analyze the antagonist specificity, efficacy, potency, mode of action, and their effects on prostate cancer cells. In collaboration with NCGC we have generated, developed, and obtained all reagents necessary for the proposed assays and tested all experimental procedures. The discovery of relaxin receptor antagonists will provide a basis for their testing as anticancer agents.